CN105925591B - Cloning and application of a key gene PeIRX10 for xylan synthesis of moso bamboo - Google Patents

Abstract

The invention belongs to the field of molecular biology and genetic engineering, and particularly relates to a key gene PeIRX10 of bamboo Glycosyltransferase (GTs) participating in xylan synthesis, wherein the gene has a nucleotide sequence shown in SEQ ID No: 1. The invention provides a powerful tool for understanding the formation of secondary cell walls and clarifying the molecular basis of the rapid growth of moso bamboo tissues; the method realizes that the PeIRX10 can be used for repairing the xylan synthesis defect in the Arabidopsis mutant and the secondary cell wall defect in the complementary Arabidopsis mutant.

Description

Cloning and application of a key gene PeIRX10 for xylan synthesis of moso bamboo

technical field

The invention belongs to the fields of molecular biology and genetic engineering, and specifically relates to the cloning and functional exploration of a key gene PeIRX10 of moso bamboo glycosyltransferases (glycosyltransferases, GTs) involved in xylan synthesis.

Background technique

Hemicellulose xylan (Xylan) is a polysaccharide component second only to cellulose in plants. It plays an important role in maintaining the stability and integrity of cell walls and is an important renewable biomass resource. Xylan is synthesized by GTs located in the Golgi. GTs are a series of enzymes involved in catalyzing the synthesis of sugar chains in disaccharides, polysaccharides, and sugar complexes. They exist in all organisms and not only participate in the synthesis of the main components of the cell wall, but also catalyze the sugar groups of various molecules and participate in development. , signal transduction, defense and other biological processes.

The IRX10 gene is a key gene involved in the elongation of the hemicellulose xylan backbone, which plays an important role in the normal thickening of the plant secondary wall. If the IRX10 gene is deleted, the xylose content in the inflorescence and stem decreases, and the cell wall appears as irregular xylem, resulting in secondary cell wall defects. At present, the research on the function of IRX10 gene in existing plants mainly focuses on model plants such as Arabidopsis thaliana and rice, but the key gene PeIRX10 involved in xylan synthesis in moso bamboo has not been reported so far.

Moso bamboo (Phyllostachys edulis) is the economical bamboo species with the largest planting area in my country, and is widely used in food, building materials, textiles, biomass energy and other fields. It has been found that the rapid growth of Phyllostachys pubescens is accompanied by secondary cell wall growth. Therefore, the identification of PeIRX10, a gene related to the synthesis of hemicellulose xylan in Moso bamboo, is of great scientific significance for understanding the formation of secondary cell walls and clarifying the molecular basis of the rapid growth of Moso bamboo tissues.

Contents of the invention

Aiming at the fact that the cause of the growth of secondary cells of Moso bamboo has not been found so far, the present invention aims to provide a new key gene PeIRX10 that is derived from Moso bamboo and participates in xylan synthesis, so as to understand the formation of secondary cell walls and clarify the rapid growth of Moso bamboo tissue. The molecular basis provides a powerful tool. The purpose of the present invention is also to provide the application of gene PeIRX10 in plant genetic transformation, so as to realize the repair of xylan synthesis defect in Arabidopsis mutant and the secondary cell wall defect in complementary Arabidopsis mutant by using gene PeIRX10.

In order to realize the purpose of the invention of the present invention, the inventor provides the following technical solutions:

The purpose of the present invention is firstly to provide a new key gene PeIRX10 involved in xylan synthesis derived from Phyllostachys pubescens, the gene has the nucleotide sequence shown in SEQ ID No:1.

As shown in accompanying drawing 1: the key gene PeIRX10 of moso bamboo xylan synthesis provided by the present invention has the highest expression level in bamboo shoots, the expression level in stems, roots and inflorescences is relatively low, and the expression level in leaves is the lowest. This expression pattern of PeIRX10 is related to the rapid growth of bamboo shoots.

Another object of the present invention is to provide the application of PeIRX10 in plant genetic transformation. On the one hand, it includes the application of the key gene PeIRX10 in the preparation of transgenic plants, and the plant is Arabidopsis; on the other hand, it includes Moso bamboo Application of PeIRX10, a key gene for xylan synthesis, in repairing xylan synthesis defects in Arabidopsis mutants and complementing secondary cell wall defects in Arabidopsis mutants. The present invention uses genetic engineering technology to recombine the PeIRX10 gene into Arabidopsis plants with irx10irx10l double mutation background, and obtain transgenic Arabidopsis plants overexpressing the PeIRX10 gene, and the complementary plant phenotype obtained by overexpressing the PeIRX10 gene is the same as that of the wild type Almost the same (as shown in Table 2 and Figure 3). Marked by the monoclonal antibody LM10, the complementary plants were detected by immunofluorescence, which showed that the xylem xylan in the cross-section of the complementary Arabidopsis stem had a strong immune signal (as shown in Figure 5), indicating that the PeIRX10 gene can repair Deficiency in xylan synthesis in Arabidopsis mutants plays an important role in xylan synthesis in Moso bamboo.

Compared with prior art, the present invention has following advantage:

1. The present invention has found a new key gene PeIRX10 that is derived from Phyllostachys edulis and participates in the synthesis of xylan, thereby providing important scientific significance for understanding the formation of secondary cell walls and clarifying the molecular basis of the rapid growth of Phyllostachys pubescens.

2. The invention uses the gene PeIRX10 to repair xylan synthesis defects in Arabidopsis mutants and complement secondary cell wall defects in Arabidopsis mutants, and plays an important role in the synthesis of xylan in Moso bamboo.

Description of drawings

Accompanying drawing 1 is the bar graph of the expression pattern analysis of PeIRX10 gene of Phyllostachys pubescens in different tissues and organs.

Accompanying drawing 2 is the RT-PCR detection of transgenic Arabidopsis plants of PeIRX10,

Among them, 1 represents wild-type Arabidopsis; 2 represents irx10irx10l double mutation homozygous plants; 3-6 represent PeIRX10 gene transgenic plants.

Accompanying drawing 3 is the phenotypic change of wild-type, double mutant plants and complementary Arabidopsis plants.

Accompanying drawing 4 is the change of the secondary cell wall of wild type, irx10irx10l double mutant and complementary plant stem.

Accompanying drawing 5 is the LM10 chemical immunological observation of the stem section of Arabidopsis thaliana overexpressing the PeIRX10 gene,

Where a represents wild-type Arabidopsis; b represents irx10irx10l double mutation homozygous plants; c represents PeIRX10 gene transgenic plants.

Detailed ways

The content of the present invention will be described in more detail below in conjunction with the embodiments and the accompanying drawings. It should be understood that the implementation of the present invention is not limited to the following examples, and any modifications and/or changes made to the present invention will fall within the protection scope of the present invention.

In the present invention, unless otherwise specified, all parts and percentages are weight units, and all equipment and raw materials can be purchased from the market or commonly used in this industry. Unless otherwise specified, the methods used in the examples are common techniques in the art.

The experimental methods for which specific conditions are not indicated in the examples are carried out according to conventional conditions, the conditions described in the Molecular Cloning Laboratory Handbook of Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989).

Example 1 Phyllostachys pubescens PeIRX10 gene expression

(1) Experimental method

1. Moso bamboo material

Moso bamboo materials of different tissues were obtained from the roots, stems, leaves, inflorescences and young shoots of 1-year-old seedlings, which were quick-frozen in liquid nitrogen and stored at -80°C for the extraction of total RNA.

(1) RNA extraction and cDNA synthesis

Perform BLAST retrieval and comparison analysis on the sequenced gene sequence database and its corresponding protein sequence database in Phyllostachys pubescens, obtain the nucleotide sequence of PeIRX10 gene (PH01004923G0080; SEQ ID No: 1), and design primers as follows:

PeIRX10-F1:5'-CCTGAACCACATGTTTGCCG-3' (SEQ ID No: 2)

PeIRX10-R1:5'-AATCGCACTGCGCATCATTC-3' (SEQ ID No: 3)

The total RNA of each sample of Phyllostachys pubescens was extracted with RNA extraction kit (OMEGA). Reverse transcription was performed using the PrimeScriptII1stStrand cDNA Synthesis Kit (Takara). The reverse transcription reaction system was: 2 μg of total RNA, 1 μL of Oligo (dT), 0.5 μL of enzyme, 0.5 μL of RNase inhibitor, 2 μL of dNTP, and ddH ₂ O to the total system is 20 μL; the reaction conditions are: 37 ° C for 15 min; 85 ° C for 5 s; the primer synthesis is carried out in Zhongmei Taihe Biotechnology (Beijing) Co., Ltd.

(2) RT-PCR analysis

The β-actin gene was selected as an internal reference, and the primers were as follows:

ACTIN-F: TGAGCTTCCTGATGGGCAAG (SEQ ID No: 4);

ACTIN-R: CCTGATATCCACGTCGCACTT (SEQ ID No: 5).

Using the cDNA obtained in step (1) as a template, PCR amplification was performed with the primers in step (1), and sequencing showed that the PeIRX10 gene (nucleotide sequence is shown in SEQ ID No: 1) was obtained. The reaction system for RT-PCR amplification is: cDNA 3 μL, 10× buffer 5 μL, LA Taq 0.5 μL, dNTP 8 μL, PeIRX10-F1 1 μL, PeIRX10-R1 1 μL, 32.5 μL ddH ₂ O, a total of 50 μL; the reaction program is: 95 ℃ for 30s; 95℃ for 5s, 60℃ for 30s, 40 cycles; the melting point curve detection program is to rise from 60℃ to 95℃ at a rate of 0.6℃/s, and read the fluorescence signal value continuously. The instrument used was CFX96 real-time fluorescent quantitative PCR instrument (Bio-Rad, USA). Each test included a negative control using H ₂ O as the reaction template. The relative quantitative △Ct method was used for the test data. Values are normalized.

(2) Experimental results

There are differences in the expression of PeIRX10 gene in different tissues and organs of Moso bamboo (see Figure 1), wherein the expression level in bamboo shoots is the highest, the expression level in stems, roots and inflorescences is relatively low, and the expression level in leaves is relatively low. lowest level.

Example 2 Overexpression of Phyllostachys pubescens PeIRX10 gene in Arabidopsis thaliana

(1) Experimental method

1. Construction of expression vector

Based on the Gateway system for PCR amplification of the full-length cDNA sequence of Phyllostachys pubescens PeIRX10, the amplification primers are:

PeIRX10-F:

5′-

GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAGGAGGTGGGTCTTGGCC-3' (SEQ ID No: 6);

PeIRX10-R:

5′-

GGGGACCACTTTGTACAAGAAAGCTGGGTCCCAAGGCTTCAGGTCGCCCACCG-3' (SEQ ID No: 7).

The PCR reaction system was 20 μL: 10 μL of 2×PrimeSTAR Max Premix, 0.5 μL of upstream and downstream primers (10 μmol L ⁻¹ ), 2 μL of template, and 7 μL of dd H ₂ O. Reaction program: pre-denaturation at 94°C for 5 minutes; 35 cycles at 94°C for 30s, 1min at 55°C for 30s, and 30s at 72°C; extension at 72°C for 10min. The amplified product was ligated with pDONR207, transformed into DH5α, and positive clones were obtained. The plasmid was extracted and identified by PCR. The reaction system: 3 μL of the recovered PCR target fragment, 1 μL of pDONR207 vector, 1 μL of BPClonase Enzyme Mix, and then sequenced. Perform LR recombination reaction with the correctly sequenced recombinant plasmid and pEarlyGate101, LR reaction system: pDONR207-PeIRX10 3 μL, pEarlyGate101 1 μL, LR Clonase Enzyme Mix 1 μL, and then transform Escherichia coli DH5α, extract the plasmid after cultivation and sequence. The recombinant plasmids with correct sequencing were transformed into Agrobacterium, and the obtained positive colonies were transformed into Arabidopsis irx10l(-/-)irx10(+/-) heterozygous plants by flower dipping method.

2. Identification of transgenic plants

Transform irx10l(-/-) irx10(-/-) double mutant Arabidopsis thaliana through genetic engineering technology, and after obtaining transgenic plants, verify by means of PCR and RT-PCR, the primers are:

IRX10-F: 5'-CCACTCGGAGGACTTGGA-3' (SEQ ID No: 8),

IRX10-R: 5'-GGAAAAAGCCATTGAAAG GG-3' (SEQ ID No: 9),

T-DNA insertion primer:

LBa1:5'-TGGTTCACGTAGTGGGCCATCG-3' (SEQ ID No: 10),

The RNA of four-week-old Arabidopsis plants of three genotypes was extracted, reverse-transcribed into cDNA, the method was as described in Example 1, the internal reference gene was the same as Example 1, and the RT-PCR reaction system and reaction procedure were the same as Example 1.

(2) Experimental results

Detected by RT-PCR, it shows that the PeIRX10 gene has been transformed into Arabidopsis thaliana and expressed (see accompanying drawing 2), in accompanying drawing 2, wherein 1 represents wild-type Arabidopsis; 2 represents irx10irx10l double mutation homozygous plant; 3- 6 is the PeIRX10 gene transgenic plant. In the background of irx10irx10l double mutant plants, the phenotype size of Arabidopsis plants complemented by overexpression of the PeIRX10 gene is almost the same as that of the wild type (see Figure 3), and has normal plant height, stem diameter, and leaf size and number (See Table 1).

Table 1 Phenotype analysis of wild type, double mutant plants and complementary Arabidopsis plants

Example 3 Cell wall xylan immunolocalization

(1) Experimental method

Take the stems at the base of wild-type, double-horn plants and complementary Arabidopsis plants that have grown for eight weeks, cut them into 1mm-thick slices, wash the slices with 0.1M phosphate buffer (pH 7.2) for 5-10min; use fresh Soak the slices in 3% skimmed milk for 1 h and blow the skim milk continuously; remove the skim milk and wash the slices with PBS buffer for 5 min; incubate the slices with rat anti-xylan antibody LM10 (Plantprobes) for 2 h, then wash the slices with PBS buffer to Wash unbound primary antibody; incubate with 50-fold diluted FITC-goat anti-rat antibody (Zomanbio, Cat.Z1319) for 2 h, then wash the section with PBS buffer 10 times to wash unbound secondary antibody; finally The slices were fixed on glass slides, observed and photographed under a laser confocal electron microscope (ZeissLSM710, 495nm), and the results are shown in Figure 4 and Figure 5 .

(2) Experimental results

1. The PeIRX10 gene can complement the secondary cell wall defect of Arabidopsis irx10irx10l double mutant plants

The irx10irx10l homozygous plants basically lacked secondary cell wall growth, and the cell walls of intervascular fibers and xylem vessel cells were significantly thinned (see Figure 4 B and E). The cell wall thickness of the inter-bundle fibers and xylem vessel cells in the PeIRX10 gene-complemented plants was almost similar to that of wild-type Arabidopsis (see Figure 4A and D). It shows that PeIRX10 gene can complement the secondary thickening defect of Arabidopsis irx10irx10l plant cell wall.

2. The PeIRX10 gene can repair the lack of xylan synthesis in the cell wall of Arabidopsis irx10irx10l double mutant plants

Table 2 Comparison of cell wall monosaccharide components in stems of wild-type, double mutant and complementary Arabidopsis plants

sample Wild type irx10irx10l irx10irx10l±PeIRX10 D 9.4±0.04Bc 11.3±0.03BCa 10.3±0.07Bb Trehalose 2.2±0.02Cb 4.5±0.05Ca 2.6±0.05Cb Arabic candy 11.1±0.03ABCc 38.6±2.12ABa 16.8±0.49ABCb xylose 102.0±5.63Aa 8.3±0.11BCc 94.1±4.96Ab Mannose 17.2±0.09ABc 20.4±3.17Ba 18.5±1.06BCb Galactose 17.6±1.08BCb 41.2±3.17ABa 19.4±3.34Aab glucose 19.84±1.98Cb 75.32±4.26Aa 26.32±2.56ABa

As can be seen from Table 2, compared with the wild type, the xylose content in the irx10irx10l double mutant plants is reduced to about 8%, while the content of other monosaccharides (such as arabinose, galactose and glucose, etc.) in the cell wall is similar to that of the wild type ratio has increased significantly. In the complementary plants, the content of xylose and other monosaccharides was similar to that of the wild type, which indicated that in the irx10irx10l mutant, the overexpression of the PeIRX10 gene could restore the monosaccharide content in the cell wall to be similar to that of the wild type.

In the cross-sections of wild-type and complemented Arabidopsis stems, the xylem has a strong immune signal, but no immune signal is detected in the irx10irx10l double mutant plants (see Figure 5), indicating that in the complementary plants with the double mutant background Overexpression of the PeIRX10 gene restored xylan deficiency in irx10irx10l double mutant plants.

Although the inventor has described and listed the technical solutions of the present invention in more detail, it should be understood that for a person skilled in the art, it is obvious to make modifications and/or modifications to the above-mentioned embodiments or to adopt equivalent alternatives. None of them can deviate from the essence of the spirit of the present invention, and the terms appearing in the present invention are used to explain and understand the technical solution of the present invention, and cannot constitute a limitation of the present invention.

Claims (3)

1. A key gene PeIRX10 for the synthesis of moso bamboo xylan, characterized in that its nucleotide sequence is as shown in SEQ ID NO:1. 2. The application of the key gene PeIRX10 in the synthesis of moso bamboo xylan according to claim 1 in the preparation of transgenic plants, characterized in that the plants are Arabidopsis thaliana. 3. The use of the key gene PeIRX10 for xylan synthesis of moso bamboo according to claim 1 in repairing xylan synthesis defects in Arabidopsis mutants and complementing secondary cell wall defects in Arabidopsis mutants.